Microscopy method and apparatus for determining the positions of emitter objects in a three-dimensional space that comprises focusing scattered light or fluorescent light emitted by an emitter object, separating the focused beam in a first and a second optical beam, directing the first and the second optical beam through a varifocal lens having an optical axis such that the first optical beam impinges on the lens along the optical axis and the second beam impinges decentralized with respect to the optical axis of the varifocal lens, simultaneously capturing a first image created by the first optical beam and a second image created by the second optical beam, and determining the relative displacement of the position of the object in the first and in the second image, wherein the relative displacement contains the information of the axial position of the object along a perpendicular direction to the image plane.
Legal claims defining the scope of protection, as filed with the USPTO.
1. Microscopy method for determining the position of an emitter object in a three dimensional (3D) space which comprises: a) illuminating an emitter object so as to cause an emission of scattered light or fluorescent light from the emitter object; b) focusing the emitted light in a primary focused optical beam through an objective lens; c) splitting the primary optical beam in a first secondary beam and in a second secondary beam; d) directing the first and the second secondary beam through a varifocal lens, having an optical axis, along respective optical paths such that the first secondary beam impinges on the varifocal lens along a direction corresponding to said optical axis and the second secondary beam impinges decentralized on the varifocal lens at an offset distance Δd from the optical axis and along a direction parallel to the same, in which the varifocal lens has an electronically controllable focal length, e) electronically controlling the focal length of the varifocal lens changing the focal length through a range of focal length values so as to move the respective focal positions of the first and second beam along said optical axis through said range of focal length values in a predetermined travel time, f) simultaneously acquiring a first image and a second image of the emitter object in an integration time greater than or equal to the travel time of the focal positions simultaneously detecting the first secondary beam in-axis and the decentralized second secondary beam in respective first detection area and second detection area arranged on an image plane (x, y); g) analyzing the first and the second image for determining a first object position on the first image and a second object position on the second image and determining a relative displacement Δr in the image plane (x, y) of the position of the object in the two images, and h) determining an axial position z p of the emitter object along an axis z perpendicular to the image plane on the basis of the relative displacement Δr.
2. Method according to claim 1 , further comprising, after step h): i) associating the coordinates defined by the first position on the image plane (x, y) and by the axial position z p with the 3D position of the emitter object.
3. Method according to claim 1 wherein, in step h), the axial position z p is determined on the basis of a linear relationship between z p and Δr.
4. Method according to claim 1 , which further comprises, subsequently to focusing the light emitted in a primary optical beam and prior to splitting the primary optical beam into a first secondary beam and into a second secondary beam, directing the primary optical beam through a relay optical unit having a magnification ratio, the relay optical unit being arranged on a rear focal plane of the objective lens.
5. Method according to claim 1 , wherein the varifocal lens is arranged on a conjugate plane of the rear focal plane of the objective lens.
6. Method according to claim 1 , wherein the offset distance Δd of the second secondary beam from the optical axis of the varifocal lens is such as to cause a lateral displacement Δr=Δy between the first and the second position of the emitter object along one of the two of the image plane coordinates (x, y).
7. Method according to claim 6 wherein, in step h), the axial position z p of the emitter object along axis z is determined according to a linear relationship Δy=C z p , wherein C is a conversion factor.
8. Method according to claim 1 , wherein the step of simultaneously acquiring a first image and a second image of the emitter object comprises simultaneously acquiring a plurality of respective first and second images at successive instants so as to trace the 3D position of the object over time.
9. Method according to claim 1 , wherein simultaneously acquiring is carried out by a two-dimensional image sensor which comprises an array of photosensitive elements which extend in the image plane (x, y) in a detection area which comprises the first detection area and the second detection area.
10. Method according to claim 1 , wherein simultaneously acquiring comprises acquiring the first secondary beam through a first two-dimensional image sensor and acquiring the second secondary beam through a second two-dimensional image sensor, wherein the first and the second two-dimensional image sensors are mutually synchronized and each image sensor comprises a respective array of photosensitive elements defining a respective first and second detection area in the image plane (x, y).
11. Method according to claim 1 , wherein: splitting the primary optical beam into a first secondary beam and a second secondary beam comprises transmitting the primary beam through a beam splitter configured for power-splitting the beam, and the beam splitter is configured in such a way as to produce a first secondary beam and a second secondary beam which propagate along two distinct directions not parallel to each other and step d) comprises directing at least one between the first and the second secondary beam through a directing system configured such that the first and the second secondary beam, in output from the directing optical system, propagate along two distinct and mutually parallel directions.
12. Microscopy apparatus for determining the position of one or more emitter objects in a three dimensional (3D) space which comprises: an objective lens configured for collecting light emitted by an emitter object and focusing the emitted light in a primary light beam; a beam splitter arranged for receiving the primary optical beam and configured for power-splitting the primary optical beam in a first secondary optical beam and a second secondary optical beam; a varifocal lens with electronically tunable focal length and having an optical axis, the varifocal lens being arranged downstream of the beam splitter; an optical beam directing optical system for directing at least one between the first secondary optical beam and the second secondary optical beam, the directing optical system being arranged between the beam splitter and the varifocal lens and configured such that the first and the second secondary beams in output from the directing optical system, propagate along two distinct and mutually parallel directions; at least one photodetector device arranged so as to receive the first and the second secondary beam in output from the varifocal lens, wherein the varifocal lens and the directing optical system are arranged in such a way that the first secondary optical beam impinges on the varifocal lens along its optical axis and the second secondary optical beam impinges on the varifocal lens decentralized along a direction parallel to the optical axis and at an offset distance Δd from the same, and the at least one photodetector device is configured for simultaneously detecting the first secondary beam and the second secondary beam on a respective first and second detection area to form at least one two-dimensional image in an image plane (x, y), which comprises respective first image of the emitter object formed on the first detection area by the first beam in axis and the second image of the same emitter object formed on the second detection area by the second decentralized beam.
13. Apparatus according to claim 12 , wherein the varifocal lens is configured to be electronically controlled by setting a variation in the focal length through a range of focal length values so as to move the respective focal positions of the first and second beam along the optical axis of the varifocal lens through said range of focal length values in a predetermined travel time and the at least one detector device is configured for forming the at least one two-dimensional image in an integration time greater than or equal to the travel time.
14. Apparatus according to claim 12 , which further comprises: a relay optical unit positioned on a rear focal plane of the objective lens ( 23 ) and arranged in such a way as to receive the primary optical beam upstream of the beam splitter, the optical relay unit being configured for transferring an image formed by the objective lens to an image plane conjugate with a magnification ratio.
15. Apparatus according to claim 14 , wherein the relay optical unit is a telecentric optical system with a magnification ratio of 1:1.
16. Apparatus according to claim 12 , wherein the varifocal lens is arranged on a conjugate plane of the rear focal plane of the objective lens.
17. Apparatus according to claim 12 , which further comprises a data processing device connected to the at least one photodetector device configured for: receiving the first image and the second image of the emitter object; analyzing the first and the second image for determining a first position of the object on the first image and a second object position on the second image, determining a relative displacement Δr in the image plane (x, y) of the position of the object in the first and in the second image; determining an axial position z p of the emitter object along an axis z perpendicular to the image plane on the basis of Δr, and associating the coordinates defined by the first position on the image plane (x, y) and by the axial position z p with the position (x, y, z) of the emitter object.
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January 16, 2018
September 15, 2020
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